Annexin V belongs to the annexin family, which is a group of proteins that are highly conserved evolutionarily, and which has the property of binding with Ca2+ and phosphate esters. They are structurally homologous and are considered to be the membrane-bound elements regulated by Ca2+. The same function of annexin family is: in the presence of Ca2+, high affinity for acidic phosphate ester molecules. The annexin family is found in the higher and lower eukaryotes. The annexin members exist in many diverse types of cells, and their biochemical effects are closely related with immune properties, such as playing a role in the anticoagulation, and anti-inflammatory process, especially in the cell replication, differentiation and exocytosis process (Iwasaki A, Suda M, Nakao H, et al, Biochem., 1987, 10: 1261; Pollard H B, Haigler H T, Biol Chem, 1990, 265: 21207). The biological function of the members of the family also need further study. Although people still do not understand very well the function in vivo of the members, however, it is thought generally that the binding activity of the annexin with phosphate is the base of annexin family's activity in vitro, and relates to their physiological properties.
At present, the main applications of annexin V are the detection of apoptosis. Apoptosis, which is also known as programmed cell death (PCD), is an important part in the cell life cycle, and is a gene-regulated initiative dead process of cells different from the process of necrosis and accidental death. Cell's apoptosis is an important way to adjust and maintain the relative balance of the organism; and is also closely related to a variety of disease pathology. In the process of development of tumors and radiotherapy and chemotherapy, cerebral and myocardial ischemia reperfusion injury and the rejection in organ transplant, the cell's apoptosis and change can be found. The cell's apoptosis is a process of programmed cell death in which cells actively participate in, involving a series of change of biological molecules and cell morphology; wherein, when the apoptosis occurs, phosphatidylserine (PS) will be flipped and exposed to the outside of the cell membrane (Fadok V A, Voelker D R, Campbell, P A, Cohen J J, Bratton D L, Henson P M, J Immunol, 1992, 148(7): 2207-2216). It is an early event in apoptosis occurring before the morphological changes of the apoptotic cells (Narula J, Strauss H W, 2003, J Nucl Med, 44(3):397-399), and it is also the initial event of apoptotic cascade reaction. As a sign of the reorganization of the apoptotic cells by phagocytes, it further lead to the shrinkage of the cytoplasm, the concentration of chromatin and the degradation of the nuclear DNA etc. Therefore, the flipped PS is the most studied and most promising and most possible application foreground detection target for the apoptosis, which can early detect the apoptosis with high timeliness, (Bold R J, Termuhlen P M, McConkey D J, Surg Oncol, 1997, 6(3):133-142). At home and abroad, it is regular to use of drugs combinations with PS-specific to detect the apoptosis.
Because of its specific affinity for (P S Meers P, Mealy T. Biochemistry, 1993, 32(43): 11711-11721) the annexin V specifically binds to the surface of a cell which is in the apoptotic process, so that, the annexin V can be used for the detection of apoptotic cells after labeled by fluorescent protein or radioactive material. This method is widely used in the research of the cell and molecular biology and immunology (Koopman G, Reutelingsperger C, Kuijten G A, et al. Blood, 1994, 84(5):1415-1420; Vermes I, Haanen C, SteVens-Nakken H, et al. J Immunol Methods, 1995, 184(1): 39-510; Zhang G, Gurtu V, Kain S R, et al. Biotechniques, 1997, 23(3): 525-531; Vriens P W, Blankenberg F G, Stoot J H, et al. J Thorac Cardiovasc Surg, 1998, 116(5): 844-853; Boldt A, Barten M J, Weiss C, et al. Cytometry A, 2006, 69(3): 158-160). The annexin V labeled by fluorescein (such as FITC, PE, etc.) or biotin was used as a probe to check the phenomenon of apoptosis through the flow cytometry or fluorescence microscopy, and that is a sensitive, efficient and mature laboratory method for detection. But the clinical specimens, in addition to blood, need be detected by tissue biopsy with traumatic, and during the operation the apoptosis is easily caused and the results of the analysis are affected. Therefore, the detection in vivo of apoptosis needs to explore new methods. For example, the apoptosis imaging in vivo with annexin V labeled by radionuclide, achieves the noninvasive apoptosis imaging of the cell in vivo, to monitor the apoptosis in vivo in real time. This type of apoptosis imaging has made great progress in the areas of monitoring cell's apoptosis in vivo. At present, the applications of the recombinant annexin V labeled with radioactive material in the assessment of ischemic injury, the allograft rejection response and the evaluation of the tumor-treatment of the chemotherapeutic drugs are still in the trial of clinical experiment (Watanabe H, Murata Y, Miura M, et al. Nucl Med Commun, 2006, 27(1): 81-89; Blankenberg F G, Kalinyak J, Liu L, et al. Eur J Nucl Med Mol Imaging, 2006, 14: 1-9).
Although the labeling of the protein of polypeptide by radioactive iodine (*I) is simple, mature, and can retain their original biological activity, but the supply of 123I and 124I fitted the imaging in vivo is difficult. The most reported is the annexin V labeled with 99mTc. The advantage of 99mTc is its half-life of 6 hours, so that a timely imaging can be obtained, and also dose to patient is less, and it can be easily obtained from Mo/Tc generator. However, the labeling of the annexin V directly by 99mTc is difficult, and the labeling rate is low, and during the labeling process there will be some denatured protein which affect its distribution of radioactivity in the body being generated (Takei T, Kuge Y, Zhao S, et al. J Nucl Med, 2004, 45(12): 2083-2087). Therefore, the labeling of the annexin V by 99mTc is based on the principle of indirectly labeling. The bifunctional chelating agents commonly used include N-1-imino-4-mercaptobutyl (Imino) (Kemerink G J, Liem I H, Hofstra L, et al. J Nucl Med, 2001, 42(2): 382-387), ethylenediamine cysteine (EC) (Yang D J, Azhdarinia A, Wu P, et al. Cancer Biother Radiopharm, 2001, 16(1):73-83), disulfur dinitride (N2S2, also known as BTAP) (Kemerink G J, Boersma H H, Thimister P W, et al. Eur J Nucl Med, 2001, 28(9):1373-1378) etc., and the most common is hydrazinonicotinyl (HYNIC), that has been clinically trialed (Penn D L, Kim C, Zhang K, et al. Nucl Med Biol, 37(1): 29-34; Rottey S, Slegers G, Van Belle S, et al. J Nucl Med, 2006, 47(11): 1813-1818; Kartachova M, Haas R L, Olmos R A, et al. Radiother Oncol, 2004, 72(3): 333-339; Kemerink G J, Liu X, Kieffer D, et al. J Nucl Med, 2003, 44(6): 947-952). Biological behaviors in vivo of the labeled products prepared by different bifunctional chelating agents has a greater difference, wherein the labeled-products prepared by 99mTc with Imino have a higher intake in the liver, kidney and spleen, and have a relatively long biological half-life in vivo; the labeled-products prepared by N2S2(BTAP) have a higher intake in the liver, kidney, spleen and inferior belly; the labeled-products prepared by HYNIC are metabolized through kidney instead of intestinal tract, so that they have a higher intake in the kidney and liver and have a long have a relatively long biological half-life in vivo (Boersma H H, Kietselaer B L, Stolk L M, et al. J Nucl Med, 2005, 46(12): 2035-2050). In addition, the indirectly labeling method is complex, and the product need being purified, and it is not easily to be made into a kit, so their clinical use is subject to certain restrictions.
The recent studies reveal that: during the genetic recombinant expression of the annexin V, after the structural modification, it becomes very profitable for the labeling directly by 99mTc, for example, Zhang Lina etc. added 10 histidines in the N-terminus of the annexin V protein (Zhang L N, Yang X, Hua Z C, Preparative Biochemistry and Biotechnology, 2000, 30(4): 305-312). A good expression the annexin V protein in which 10 histidines are added is obtained, and it has high activity for the detection of apoptosis in vivo and vitro (Zhang L N, Yang X, Hua Z C, Preparative Biochemistry and Biotechnology, 2000, 30(4): 305-312; Ye F, Fang W, Wang F, et al. Nuclear Medicine and Biology, doi: 10.1016/j.nucmedbio.2010.11.002; Zheng Yumin, Wang Zizheng, Yan Jue, etc. Journal of Chinese nuclear medicine, 2008, 28(6): 378-382; Song Liping, Hua Zichun, Zhang Xin, etc. Journal of China clinic medical imaging, 2010, 21(1): 53-55; Song Liping, Hua Zichun, Zhang Xin, etc. Journal of China clinic medical imaging, 2010, 21(5): 358-360). Tait J F, etc. added 7 amino acids in the N-terminus, including 1-2 cysteine residues, to get three mutants, separately named as: annexin V-116, annexin V-117 and annexin V-118 (Tait J F, Brown D S, Gibson D F, et al. Bioconjug Chem, 2000, 11(6): 918-925; Tait J F, Smith C, Gibson D F, Bioconjug Chem, 2002, 13(5): 1119-1123). Their binding activitis with membrane are consistent with the natural annexin V, and have similar biological distributions with the 99mTc-HYNIC-annexin V (Tai J F, Brown B S, U.S. Pat. No. 7,204,972 B2). The three variants of the annexin V can express in the cytoplasm of the E. coli, and can be isolated and purified, with the product yield of 10 mg/L (Tait J F, Brown D S, Gibson D F, et al. Bioconjug Chem, 2000, 11(6): 918-925; Tait J F, Smith C, Gibson D F, Bioconjug Chem, 2002, 13(5): 1119-1123; Tai J F, Brown B S, U.S. Pat. No. 7,204,972 B2). However, the application of the recombinant annexin V has the following questions:
Currently, recombinant annexin V labeled with radioactive material has the disadvantages of low labeling efficiency. Meanwhile, due to the low labeling rate and the generation of some denatured proteins occurring during the labeling, the radioactive distribution of the annexin V in vivo will be affected. In clinically trials, it is also found that the annexin V is mainly distributed in kidney, liver and spleen (Rottey S, Van den Bossche B, Slegers G, Van Belle S, van de Wiele C, Q. J. Nucl. Med. Mol. Imaging, 2009, 53(2): 127-32).
Because of the low yield, the preparation method of the recombinant annexin V does not meet the needs of practical application. Due to the recombinant annexin V commonly is the form of inclusion body when express in E. coli, so the foreign scholars researched the recombinant annexin V expressed by the yeast to solve the problem of easy formation of inclusion bodies in E. coli. Even in the latest research and patents of the international authority on annexin V, American professor Tait J F and his research group, three kinds of recombinant annexin V variant, in which N-terminus seven amino acids are added, are expressed in the in E. coli and purified, its productivity is only 10 mg/L, and the needs of the practical application is meet unsuccessfully. The purification process including ultrasonication, cell membrane adsorption and dissociation, Mono Q ion exchange chromatography (elution concentration: 0.22 M NaCl), ultrafiltration and dialysis. The inventor of the present invention built an annexin V in which 10 histidines are added in the preliminary studies 10 years ago, and the labeling efficiency significantly increased and method is simple, but the final productivity was only 7.4 mg/L (Zhang L N, Yang X, Hua Z C, Preparative Biochemistry and Biotechnology, 2000, 30(4): 305-312). The expression level and yield is similar to the yield of the three recombinant variants of the annexin Vin which N-terminus seven amino acids are added by American professor Tait J F and his research group, that indicate that: there is a exist serious bottleneck in the production of recombinant annexin V.